Abstract Hematopoietic stem cells (HSCs) are capable of extensive self-renewal and multi-lineage differentiation to maintain lifelong production of all blood cells. However, it is well documented that the self-renewal capacity of HSCs declines with age and old HSCs exhibit a skewed differentiation towards myeloid lineages at the expense of lymphopoiesis. These age-associated changes are responsible for decreased immunity and increased propensity for anemia and myeloid malignancies in the elderly. HSCs undergo complex changes in gene expression during aging. Whether these changes are responsible for HSC aging, and more importantly whether modulation of any of these changes can rejuvenate old HSCs is largely unknown and unexplored. We hypothesize that certain genes whose expression is up-regulated in HSCs during aging represent the ?aging? factors and are responsible for the age-dependent decline in HSC self-renewal and function, whereas a class of down-regulated genes during HSC aging are the ?fountain of youth? type of factors with their decreased expression leading to HSC aging. We further hypothesize that depletion of critical ?aging? genes will rejuvenate old HSCs. To test these hypotheses, we will systematically identify genes that regulate HSC self-renewal and aging using an unbiased CRISPR-based in vivo genetic screen with the following two aims. In Aim 1, we will use a focused lentiviral CRISPR library to deliver the Cas9 nuclease and 21,268 single-guide RNAs (sgRNAs) to target 5,317 mouse genes that show altered expression in HSCs during aging. HSCs isolated from young mice will be infected with this CRISPR library and infected HSCs that carry different sgRNAs will be injected into stem cell-depleted recipient mice to repopulate competitively with each other in two rounds of transplantation. Altered HSC self-renewal and function will lead to either enrichment or depletion of HSCs. By analyzing the abundance of specific sgRNAs in HSCs and differentiated B, T or myeloid lineages using deep sequencing before and after transplantations, we will identify the corresponding genes that positively or negatively regulate HSC self- renewal and differentiation. In Aim 2, we will validate selected candidate genes identified in Aim 1 based on their significant enrichment or depletion in the screen and determine whether the processes affected by these candidate genes involve HSC homing, proliferation, quiescence or differentiation. Furthermore, we will test whether deletion in old HSCs of candidate ?aging? genes and four genes (Gadd45a, Gadd45g, Runx1 and Selp) that have been shown to negatively regulate HSC self-renewal in young mice can enhance HSC self-renewal, reverse the skewed differentiation and thus rejuvenate old HSCs. The proposed studies will provide novel insight into the underlying mechanisms of HSC self-renewal, differentiation and aging. The translational potential of HSC rejuvenation discovered in the proposed studies will establish a paradigm for ameliorating age-associated diseases and promoting healthspan.